Abstract

Development requires the proper execution and regulation of the cell cycle, via precise, conserved mechanisms. Critically, the E2F/DP complex controls the expression of essential genes during cell cycle transitions. Here, we discovered the molecular function of the Arabidopsis thaliana SUMO E3 ligase METHYL METHANESULFONATE SENSITIVITY GENE21 (AtMMS21) in regulating the cell cycle via the E2Fa/DPa pathway. DPa was identified as an AtMMS21-interacting protein and AtMMS21 competes with E2Fa for interaction with DPa. Moreover, DPa is a substrate for SUMOylation mediated by AtMMS21, and this SUMOylation enhances the dissociation of the E2Fa/DPa complex. AtMMS21 also affects the subcellular localization of E2Fa/DPa. The E2Fa/DPa target genes are upregulated in the root of mms21-1 and mms21-1 mutants showed increased endoreplication. Overexpression of DPa affected the root development of mms21-1 and overexpression of AtMMS21 completely recovered the abnormal phenotypes of 35S:E2Fa-DPa plants. Our results suggest that AtMMS21 dissociates the E2Fa/DPa complex via competition and SUMOylation in the regulation of plant cell cycle.

The Phenotype of mms21-1.(A) The phenotypes of 1-week-old (left panel) and 3-week-old (right panel) wild-type and mms21-1 plants. Bar = 1 cm.(B) The endoreplication levels in wild-type and mms21-1 plants were quantitatively analyzed by flow cytometry. The cotyledons or the first pair of true leaves (leaf 1-2) of 13-d-old plants were collected for analysis. The data are means ± sd from at least three biological replicates.

DPa Interacts with AtMMS21.(A) The interaction between DPa and AtMMS21 was confirmed in a yeast two-hybrid assay.(B) Identification of the domain on DPa required for its interaction with AtMMS21 by yeast two-hybrid analysis. The DNA binding domain (red) and dimerization domain (blue) of DPa and the SP-RING domain (purple) of AtMMS21 are indicated.(C) Identification of the domain on AtMMS21 required for its interaction with DPa by yeast two-hybrid analysis.(D) The interaction between rice DP and MMS21 in a yeast two-hybrid assay.(E) The interaction between DPa and AtMMS21 in an in vitro pull-down assay. The DPa-FLAG proteins were incubated with immobilized GST or GST-AtMMS21, and proteins immunoprecipitated with glutathione sepharose were detected using anti-FLAG antibody. The amounts of GST and GST-AtMMS21 are shown in the bottom panel.(F) The subcellular colocalization of DPa and AtMMS21. CFP-AtMMS21 and YFP-DPa were coexpressed in protoplasts. After 48 h, the CFP and YFP signals were detected and merged. Bar = 10 µm.(G) The interaction between DPa and AtMMS21 in an in vivo coimmunoprecipitation assay. Total protein extracts from transgenic plants carrying 35S:DPa-FLAG alone or both 35S:DPa-FLAG and 35S:MYC-AtMMS21 were immunoprecipitated with the immobilized anti-MYC antibody. The proteins from crude lysates (left) and immunoprecipitated proteins (right) were detected using anti-FLAG or anti-MYC antibody.

AtMMS21 Enhances the SUMOylation of DPa and Interferes with the Interaction between E2Fa and DPa.(A) The effects of AtMMS21 on the interaction between DPa and E2Fa in a yeast three-hybrid assay. Yeast cells were transformed with pBridge-BD-DPa-ProMET-AtMMS21 or pBridge-BD-DPa with pGADT7-E2Fa and cultured on SD-L-W-M medium. The relative α-galactosidase activities are shown. The activity from the sample with BD-DPa and AD-E2Fa was set to 1. The data are means ± sd from three independent experiments. ***P < 0.001, Student’s t test.(B) The effects of AtMMS21 on the DPa–E2Fa interaction in plant cells. YFP-E2Fa was expressed in protoplasts from wild-type or transgenic plants overexpressing DPa-FLAG alone or both DPa-FLAG and MYC-AtMMS21. The interaction was detected by immunoprecipitation on anti-FLAG resin. The total lysates (input) and the immunoprecipitated proteins (IP) were detected with an antibody recognizing YFP. The expression levels of DPa-FLAG and MYC-AtMMS21 were also detected using the indicated antibodies.(C) The SUMO conjugation of DPa was detected in a reconstituted SUMOylation system in E. coli. In the presence of E1 and SUMO1, the unconjugated and SUMO-conjugated wild type or the mutant with K139/140/146R substitutions of FLAG-tagged DPa with or without E2 were detected with anti-FLAG antibody.(D) AtMMS21 mediates the in vitro SUMOylation of DPa. Affinity-purified DPa-FLAG was used as a substrate in an in vitro SUMOylation assay. The SUMOylation levels of DPa in different samples were detected in an immunoblot using anti-FLAG antibody. Asterisk indicates an unspecific signal.(E) The positions of SUMO attachment sites on the DPa protein. The three-dimensional structure of the C terminus of Arabidopsis DPa was predicted using Swiss Model. The potential SUMOylation sites are indicated.(F) The effect of SUMO on the interaction between DPa and E2Fa. SUMO1 was fused to DPa-C (144 to 292 amino acids) in BD vector and used in a yeast two-hybrid assay with AD-E2Fa to detect their interaction. BD-DPa-C without SUMO was used as a control.

AtMMS21 Affects the Translocation of E2Fa/DPa.(A) Subcellular distribution of GFP-fused DPa proteins in protoplasts. Plasmids carrying 35S:DPa-GFP alone, or both 35S:DPa-GFP and 35S:AtMMS21, were transfected into wild-type protoplasts with or without CFP-E2Fa. When coexpressed with CFP-E2Fa, only the protoplasts with both CFP and GFP signals were used for analysis. Representative GFP signals from the majority of protoplasts from DPa-GFP alone, a combination of DPa-GFP and CFP-E2Fa, and a combination of DPa-GFP, AtMMS21, and CFP-E2Fa are shown. The autofluorescence from chloroplasts and bright-field (BF) signals were also detected and merged. Statistical data from means ± sd from three independent biological replicates (n > 100) are shown in the right panel. Bar = 10 µm.(B) Subcellular distribution of YFP-fused E2Fa in protoplasts. YFP-E2Fa was transiently expressed in protoplasts from wild-type and transgenic plants carrying 35S:DPa alone or both 35S:DPa and 35S:AtMMS21. Representative YFP signals from the majority of the indicated protoplasts are shown. Statistical data from means ± sd from three independent biological replicates (n > 100) are shown in the right panel. ***P < 0.001, Student’s t test. Bar = 10 µm.

Overexpression of DPa Affects Root Development in mms21-1.(A) The relative expression levels of E2Fa target genes in the roots of 4-d-old seedlings were analyzed by real-time PCR. The expression level in the wild type was set to 1. The data are means ± sd from triplicate experiments. *P < 0.05, **P < 0.01, Student’s t test.(B) The phenotypes of mms21-1 and mms21-1×35S:DPa plants. The photograph was taken 15 d after germination. Statistical data of root length shown in the right panel are means ± sd from at least 30 seedlings in three biological independent experiments. **P < 0.01, Student’s t test. Bar = 1 cm.(C) Representative root apical meristems from wild-type, 35S:DPa, mms21-1, and mms21-1×35S:DPa plants. Roots from 5-d-old seedlings were stained with . The meristem region is indicated by arrowheads; quiescent center is indicated by the lower arrowhead. Bar = 100 µm.(D) The relative expression levels of E2Fa target genes in the protoplasts of mms21-1 and mms21-1×35S:DPa with or without RNA interference for E2Fa. The protoplasts were collected 60 h after transformation and used for RNA extraction for real time PCR. The expression level in mms21-1 with empty vector was set to 1. The data are means ± sd from three experiments. *P < 0.05, ** P < 0.01, Student’s t test.

AtMMS21 Interferes with the Function of the E2Fa/DPa Complex in Plants.(A) The phenotypes of the wild type, 35S: E2Fa-DPa, and three independent lines (#1, #2, and #3) from verified 35S:DPa-E2Fa-AtMMS21 plants. The photograph was taken 7 d after germination. Bar = 1 cm.(B) Statistical data for root lengths of the indicated plants. The results are means ± sd from at least 35 seedlings per line at 7 DAG in three independent experiments. ***P < 0.001, Student’s t test.(C) The phenotypes of 3-week-old seedlings.(D) The endoreplication levels in the indicated plants were quantitatively analyzed by flow cytometry. The cotyledons of 8-d-old seedlings were used for analysis. The results are means ± sd from of at least three biological replicates.(E) The relative expression levels of E2Fa target genes in the indicated plants were analyzed by real-time PCR. The expression level in the wild type was set to 1. The data are means ± sd from triplicate experiments. **P < 0.01, ***P < 0.001, Student’s t test.(F) The effect of mutated AtMMS21 on the function of E2Fa/DPa. The root length of two independent lines (#1 and #2) of 35S:E2Fa-DPa-AtMMS21mut was compared with that of the wild type, 35S:E2Fa-DPa, or 35S:E2Fa-DPa-AtMMS21. The photograph was taken at 7 d after germination, and the statistical data are means ± sd from at least 20 seedlings. ***P < 0.001, Student’s t test. Bar = 1 cm.

Predicted Model of the Role of AtMMS21 in Plant Cell Cycle Regulation.(A) The effect of physical competition and SUMOylation on the E2Fa/DPa complex.(B) The role of AtMMS21 in cell cycle checkpoint regulation.